This invention relates generally to a medium for the in vitro culture of mammalian cells More specifically, the invention is a defined basal nutrient medium for serum-free culture or for culture when supplemented with low levels of serum. For serum-free culture, inorganic iron sources and/or defined proteins are added to the basal nutrient medium The medium is very effective when used in either high or low density culture of a wide variety of cell lines and cell types.
For in vitro culture, a medium must, of course, supply all essential nutrients for the cells: vitamins, amino acids, lipids, nucleic acid precursors, carbohydrates, trace elements, and bulk ions. Historically, basal nutrient media were designed to support cell growth only after being supplemented with a biological fluid or extract, e.g., serum or other blood products (such as plasma, plasma proteins, hemoglobin) lipids, yeast extracts or embryo extracts. Serum, in particular, proved to be an effective supplement, presumably because it contains the necessary growth- and multiplication-promoting factors in physiologically acceptable concentrations. Examples of basal nutrient media of this type are Eagle's basal medium (BME), the composition of which is recited in U.S. Pat. No. 3,450,598 (Welsh et al.), and Dulbecco's Modified Eagle's (DME) medium, the composition of which is recited in Table II of Ham et al., "Media and Growth Requirements," Methods of Enzymology, (1978). DME medium, which contains relatively high concentrations of the essential amino acids and sugars, is representative of the commercially available media formulated for the mass culture of cells with serum supplementation.
With growing sophistication in cell culture techniques, factors present in serum or other biological extracts have been identified. It is now possible to grow mammalian cells in a serum-free environment, by supplementing a basal nutrient medium with defined proteins necessary for cell growth and multiplication. For example, Ham's F12 medium was formulated for clonal cell growth. F12, the composition of which is given in Table II of Ham et al., supra, contains low concentrations of the essential amino acids and sugars, but includes lipids, nucleic acid derivatives, vitamins and nonessential amino acids.
It is now generally accepted that a readily obtainable and sufficiently complex basal nutrient medium for mass culture of cells in low serum concentrations can be fabricated by mixing DME and F12 media. Such mixtures, when supplemented with the appropriate protein factors, can also support the serum-free growth of many cell types. Barnes et al., "Methods for Growth of Cultured Cells in Serum-Free Medium," Analytical Biochem., Vol. 102, pp. 255-270 (1980), describes examples of both approaches.
Several commercially available nutrient media are based on mixtures of DME, F12 and/or other media such as those listed in Table II of Ham et al., supra. However, simple mixtures of existing commercial media are by no means optimal for culturing all cell lines and medium preparations therefore have been targeted largely to particular cell lines or cell types. Wolfe et al., "Continuous Culture of Rat C6 Glioma in Serum-Free Medium," J. Cell Biol., Vol. 87, pp. 434-41 (1980), teaches the use of a 3:1 DME-to-F12 mixture, supplemented with trace elements, and further supplemented with the following defined proteins: insulin, transferrin, fibroblast growth factor, linoleic acid complexed to fatty acid-free bovine serum albumin, and serum-spreading factor (vitronectin).
With the increasing use of cultured mammalian cells to produce biologicals (e.g., monoclonal antibodies and genetically engineered proteins), there is an increasing demand for chemically defined, serum-free media. Purification of the desired cellular product is greatly complicated by the presence of serum, which may contain at least several hundred different proteins. It is therefore desired to reduce the protein content of the culture medium to a few defined compounds from which the monoclonal antibody or other cellular product can be separated more readily. Defined protein includes any specific, identifiable, purified protein. Defined proteins include, but are not limited to, albumin, transferrin, insulin, vitronectin, fibroblast growth factor, insulin-like growth factor, laminin, fibronectin and its derivatives, and other hormones, growth and attachment factors.
In addition to necessary nutrients and protein factors, the medium must have a means for controlling pH levels. Most typically, pH control relies on a bicarbonate/carbon dioxide buffer system, which requires carbon dioxide regulators as well as incubators which supply a constant level of carbon dioxide to the culture. The buffering capacity of the system can be expanded by the inclusion of a biocompatible organic buffer, such as alpha-glycerolphosphate or HEPES (N-2-hydroxy-ethylpiperazine-N'-2-ethanesulfonic acid) or other zwitterionic buffers. Alternatives to the cumbersome bicarbonate/carbon dioxide buffer system have been proposed, but have not received significant acceptance in the field. See, for example, Leibovitz, "The Growth and Maintenance of Tissue-Cell Cultures in Free Gas Exchange with the Atmosphere," Am. J. Hygiene, Vol. 78, pp. 173-80 (1963), disclosing a medium (L-15) which uses free base amino acids and substitutes D(+)galactose, sodium pyruvate and DL-alpha-alanine for glucose. It is taught that the L-15 medium can be used in free gas exchange with the atmosphere. Ferguson, et al. (Analyt. Biochem (1980) 04:300-310) describe using zwitterionic buffers in mammalian tissue culture However, in every case the media was also supplemented with 10% serum, incubated in air containing 5% CO.sub.2, and supported cells only at low density. Bhattacharyya et al. (Gamete Research (1988) 19:123-129) used synthetic organic buffers in culture medium as a replacement for bicarbonate/carbon dioxide. Their research indicated that bicarbonate/carbon dioxide is far better in supporting the production and growth of embryos in in vitro fertilization than any of the synthetic organic buffers. To date, no media have been formulated specifically to support growth at high and low cell densities in the absence of serum and carbon dioxide incubation.